Preparation of microorganisms comprising omega-3-fatty acid for use as a prophylactic or therapeutic agent against parasitic diseases of animals

ABSTRACT

Preparation of microorganisms comprising omega-3-fatty acids for use as a prophylactic or therapeutic agent against parasitic diseases of animals 
     The present invention relates to the use of a preparation of microorganisms containing omega-3-fatty acid for preparing a medicament for prophylactic and therapeutic use against a parasitic disease of animals.

DESCRIPTION

Preparation of microorganisms comprising omega-3-fatty acids for use asa prophylactic or therapeutic agent against parasitic diseases ofanimals

The present invention relates to the use of a preparation containingomega-3-fatty acid for preparing a medicament for prophylactic andtherapeutic use against a parasitic disease of animals.

Omega-3-fatty acids (omega-3-polyunsaturated fatty acids=omega-3-PUFAs),in particular EPA (eicosapentaenoic acid) and DHA (docosahexaenoicacid), have recently become more and more important for the human diet.They have positive dietary and health-enhancing effects. With respect tolowering the cholesterol level and reducing the frequency ofcardiovascular disorders, it is sensible to enrich the human diet withomega-3-fatty acids. The positive effect of omega-3-PUFAs onpathological metabolic processes has been demonstrated in a large numberof studies. Moreover, docosahexaenoic acid plays an important role as abuilding block in the central nervous system (brain) and in the retina.

Hitherto, except for fish breeding, there have been relatively fewinvestigations into the significance of omega-3-PUFAs in the field ofanimal health. Up to now, omega-3-PUFA preparations have been primarilyadministered to animals via the feed with the intention of enrichingcertain animal products (eggs, meat) intended for human consumption withomega-3-PUFAs. Thus, the omega-3-PUFA content of the fatty tissues ofthe muscles of poulards could be increased significantly by feeding thema PUFA-containing product (Mooney & van Elswyk, 1995, Poultry Science74, Suppl. 1, 89). Furthermore, it was shown that it is possible toenrich eggs with omega-3-PUFAs by enriching the feed of laying hens withomega-3-PUFA-containing components (linseed oil, rapeseed oil, soya oil,fish oil) (Herber & van Elswyk, 1995, Poultry Science 74, Suppl. 1, S.57;

Grashorn and Blanch 1996, DGS 6, 6-9).

Hitherto, there have been no investigations into the use of omega-3-PUFApreparations in the context of prophylactic and therapeutic measures forcontrolling parasitic diseases of useful animals. In modern poultryfarming, coccidiosis caused by a certain protozoan species (orderEimeria) has great practical economical importance as an intestinaldisease, owing to the high proportion of losses. In a study which hasrecently been published, it was shown that the course of a coccidiainfection can be influenced positively by enriching the feed with fishoil (reduction in the number/intensity of the lesions in the intestine,reduction in weight loss) (Muirhead, 1995, Feedstuffs, Nov. 6 1995, 12).This effect was ascribed to the action of the omega-3-PUFAs present infish oil.

Worm infections are widespread throughout the world among agriculturallyuseful animals and lead to economic losses, in particular among younganimals.

Omega-3-PUFA preparations develop, in the digestive tract, an activitydirected against helminths (worms) which parasitize there.

However, the use of fish oil as a source of omega-3-fatty acids is notunproblematic since, in addition to a number of other fatty acids,cholesterol and certain heavy metals are also present in fish oil.Moreover, the composition of the PUFA content varies, in some casesconsiderably, depending on the kind of fish, the season and the fishingarea, which makes the preparation of PUFA products with a uniformconcentration content from fish difficult. Furthermore, fish oil can beemployed in the feed diets only in a limited amount, owing to the effecton the end products in terms of taste and smell. A further problem whichhas to be emphasized is the fact that the omega-3-PUFA concentration infish oil is usually at most 15-20%, and it is technically very difficultto concentrate the fatty acids, which is necessary for preparing a feedadditive.

The present invention solves the abovementioned problems by usingaquatic microorganisms (microalgae, protozoa, fungi, bacteria) asproduction strains for preparing preparations comprising omega-3-PUFAs,in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA). These preparations are employed as feed additives to developtheir antiparasitic (in particular coccidiostatic) effect. Theproduction strains are cultivated heterotrophically or autotrophically,harvested and subsequently dried and/or extracted. The PUFA preparationscan be employed here in the form of biomass or oil as feed additive.

Consequently, the present invention specifically relates to the use of apreparation containing omega-3-fatty acid which is obtainable fromaquatic microorganisms for preparing a medicament for prophylactic ortherapeutic use against a parasitic disease of animals.

The preparation preferably comprises the biomass which is obtainable bycultivation of aquatic microorganisms, is present in the form of an oil,or both.

According to a particularly preferred embodiment of the presentinvention, the preparation containing omega-3-fatty acid is obtainablefrom heterotrophically cultivable microalgae, and these microalgaeparticularly preferably belong to the order Crypthecodinium or the orderEuglena.

In a further preferred embodiment, the abovementioned preparation isobtainable from lower fungi, preferably of the orders Thraustochytriumor Mortierella.

In a further preferred embodiment of the present invention, the aquaticmicroorganisms belong to the photosynthesizing microalgae, which arepreferably selected from the orders Phaeodactylum, Isochrysis, Monodus,Porphyridium, Spirulina, Chlorella, Botryococcus, Cyclotella, Nitzschia,Dunaliella and Nannochloropsis.

A further preferred embodiment provides that the abovementionedpreparation is obtainable from bacteria, preferably selected from theorders Alteromonas and Shewanella.

The attached FIGURE, which forms part of this application, contains thegraphic evaluation of the excretion of oocysts by chicken in a controlgroup without administration of PUFA and in a group to which PUFA wasadministered.

Alternatively, the preparation is obtainable from mixtures of two ormore different ones of the abovementioned aquatic microorganisms.

The preparation containing omega-3-fatty acid is particularly suitablefor preparing a medicament for prophylactic and therapeutic use againstavian coccidiosis.

The abovementioned preparation is furthermore also suitable forpreparing a medicament for prophylaxis or therapy of an endoparasiticdisease of large or small animals.

Consequently, the present invention also relates to a medicament forprophylactic or therapeutic use against a parasitic disease of animalswhich comprises an omega-3-fatty-acid-containing preparation which isobtainable as described above.

The medicament can also be combined with one or more other medicaments.

The medicament according to the invention for the therapy andprophylaxis of coccidiosis is particularly effective if it contains atleast one further coccidiostat, preferably salinomycin.

The present invention will now be described in detail below.

Particularly suitable producers for preparing theomega-3-fatty-acid-containing preparation are heterotrophically growingmicroalgae and protozoa, such as Crypthecodinium and Euglena; lowerfungi, such as Thraustochytrium, Schizochytrium and Mortierella;photosynthesizing microalgae of the orders Phaeodactylum, lsochrysis,Monodus, Porphyridium, Spirulina, Chlorella, Botryococcus, Cyclotella,Nitzschia, Dunaleilla and Nannochloropsis and bacteria, for example ofthe orders Alteromonas and Shewanella.

The preparation process of the omega-3-fatty-acid-containing preparationcomprises the following steps:

inoculation of the culture medium with the producer strain

incubation of the culture under suitable conditions, it being possiblefor the cultivation to be carried out as a batch, feed-batch orcontinuous fermentation.

harvest of the culture using, inter alia, the following methods:cryopelletization, filtration, centrifugation, spray-drying.

if the biomass is not directly used as omega-3-PUFA preparation, anextraction step (for example supercritical CO₂ extraction or extractionwith organic solvents) to obtain an extract may follow, using either themoist or the dried biomass.

The preparation in the form of an extract may optionally be purifiedprior to use, for example by SFC (supercritical fluid chromatography) orHPLC methods.

In the preparation according to the invention, the omega-3-PUFAs may bepresent, inter alia, in the form of phospholipids, glycolipids, mono-,di- or triglycerides or sulfolipids, or else as free acids or ethylesters thereof.

The DHA or EPA content of the omega-3-PUFA preparation (biomass or oil)which is admixed to the feed is between 5 and 80%, based on the totalfatty acid content.

The oil may furthermore be microencapsulated before use, to ensurebetter processing and a protection of the PUFAs. To increase theoxidative stability of the omega-3-PUFAs, it is possible to addstabilizers such as, for example, tocopherol and ascorbyl palmitate tothe preparation.

The coccidiostatic action of the omega-3-fatty-acid-containingpreparation is controlled using infection experiments with the targetspecies poulards. The infective species used are Eimeria acervulina, E.maxima and E. tenella. The experiments are conducted for a period of 3weeks. In the experiments, the effect of the PUFAs on the course of theinfection is monitored at two different concentrations.

Administration and treatment plan:

    ______________________________________                                                               Number                                                   Treatment groups Preparation of animals Dosage Infection                    ______________________________________                                        A    Control     --        15     --    --                                      B Treatment yes 15 dose 1 --                                                  C Treatment yes 15 dose 2 --                                                  D Control -- 15 -- Yes                                                        E Treatment yes 15 dose 1 Yes                                                 F Treatment yes 15 dose 2 Yes                                               ______________________________________                                    

Various parameters are monitored during the studies. The followingparameters indicate the effectiveness of the PUFAs:

individual development of the weight of the animals

oocyst excretion

reduction of the lesions in the intestine (registered using the lesionscore index)

mortality rate

Furthermore, it is possible to control the action of the coccidiostatsalinomycin in combination with the omega-3-fatty-acid-containingpreparation according to the present invention using sensitivity tests.

The damage to adult worms (therapeutic effect) and to developing wormstages (prophylactic effect) in a medium containing the preparationaccording to the invention can be tested in suitable in vitro testmodels using worms which do not live parasitically (for exampleCaenorhabditis elegans), or using exogenus stages of parasitic wormswhich develop in the environment (for example trichostrongylidae of theruminants). The criteria used for the effectiveness of the preparationaccording to the invention are survival and mobility of the worms, andthe rate of development of the juvenile stages.

Test

The effect of the combination PUFA (docosahexaenoic acid fromCrypthecodinium) and salinomycin on the course of the infection afterinfection with E. tenella (Houghton, laboratory strain) was studied,with particular consideration of the individual total oocyst excretionin a feeding experiment with poulards.

Feeding a corn-rich diet (percentage of corn 44.6%), the effect of thecombination docosahexaenoic acid/salinomycin on the Eimeria infectionwas studied in the experiment. To be able to draw a conclusion on theeffect of the combination on the pathogenicity of the causativeorganism, the total oocyst excretion and the extent of the lesion scoresin the caeca of each test animal were determined. The weight developmentand the feed conversion of the animals gave further indications on theextent of the infection.

    ______________________________________                                        Characteristics of the study                                                    Test period: 20 days                                                        ______________________________________                                        Test preparations                                                               Name: Sacox ®  RBD-DHASCO ®                                             docosahexaenoic acid                                                        Concentration: Active compound contains 40% docosa-                            salinomycin hexanoic acid (DHA)                                              Manufacturer: HRVET Martek Biosciences                                          Corporation, USA                                                            Test system                                                                   Species: Lohmann fattening chicken                                            Sex: Male                                                                     Age: One-day old chicken                                                    ______________________________________                                    

    ______________________________________                                        Test phases                                                                       Test phase             Day of test                                        ______________________________________                                        Arrival of the animals/test start                                                                    d - 8                                                    Division into groups                                                          Group feces control for foreign infections d - 3                              Labeling of the animals (poultry tags)                                        Start of separate housing                                                     Infection of the animals (challenge) d0                                       Group feces control for foreign infections d + 3                              Group feces collection (control groups) d + 4 to d + 10                       Individual feces collection (infection groups)                                Determination of the total oocyst output                                      Determination of the lesion score d + 11                                    ______________________________________                                    

    ______________________________________                                        Administration and treatment scheme                                           ______________________________________                                        Administration of Sacox ® + RBD-DHASCO ®:                                                     orally with the feed                                    Infection with E. tenella, Houghton orally by means of                        (laboratory strain): an oesophageal tube                                      Infection dose: 500 sporolated                                                 oocysts/animal                                                                (in 1 ml of 1% w/v                                                            aqueous ox bile                                                               solution)                                                                    Age of the oocysts at infection: 21 days                                    ______________________________________                                    

    ______________________________________                                        Groups                                                                              Name/feed ration                                                                            Preparation                                                                             n   Dosage                                                                              Infection                             ______________________________________                                        A     control/com diet                                                                            --        15  --    --                                      B Sacox salinomycin 15 60 ppm --                                              C Sacox/RBD-DHASCO salin/DHA 15 60 ppm/ --                                        4%                                                                        D control/corn diet -- 15 -- yes                                              E Sacox salinomycin 15 60 ppm yes                                             F Sacox/RBD-DHASCO salin/DHA 15 60 ppm/ yes                                       4%                                                                      ______________________________________                                    

Work-up of the sample material

Feces collection

The feces were collected daily over a period of 24 hours in plasticbowls containing 4% strength potassium dichromate solution. All thefeces were transferred to a collection vessel.

Work-up of the feces samples

The individual feces samples were stirred (magnetic stirrer, 250 rpm),sieved (mesh size 1 mm) and made up with conc. NaCl solution to a finalvolume of 300 or 800 ml (depending on the expected oocyst excretion).Subsequently, an excess of crystalline NaCl was added with stirring.During stirring, approximately 1 ml of the suspension (from the middleof the liquid column) was sampled using a pipette or a syringe, and theMcMaster chamber was filled with this sample.

Counting of the oocysts

From the side facing the investigator, the chambers were filledbubble-free. Prior to filling, the cover glass was shifted such that anapproximately 3 mm wide gap was formed above the chambers. Beforecounting, the cover glass was moved into its proper position (thechambers have to be covered completely). After flotation for about 5minutes, the oocysts were counted in the 10 sections of the 3 countingnets, at a magnification of 80 or 320.

Taking into account the volume under each counting net (10 mm×10 mm×1.5mm=0.15 ml) and the volume of the suspension, the number of oocysts wascalculated.

Practice of the experiment

The entire experiment was carried out according to the principle of goodlaboratory practice.

The results are shown in the table below and in the FIGURE.

    __________________________________________________________________________    Weight development, feed intake, feed conversion and oocyst excretion                  Life weight                                                                             Feed        Oocyst                                                  Start                                                                            End                                                                              Increase                                                                          intake                                                                             conversion                                                                           excretion ×                                g g g/d g/d kgFl/kgLWI* 1000/animals                                        __________________________________________________________________________    A        102                                                                              580                                                                              34  57   1.6                                                     control 100.0 100.0 100.0 100.0 100.0                                         B 103 566 33 57 1.6                                                           Sacox 60 ppm 101.0 97.6 97.0 100.0 100.0                                      C 100 571 34 56 1.5                                                           Sacox 60 ppm + 98.0 98.4 100.0 98.2 93.8                                      RBD-DHASCO 4%                                                                 D 102 560 33 56 1.6 62,795                                                    control 100.0 96.6 97.0 98.2 100.0 100.0                                      Infection group                                                               E 103 551 32 56 1.6 6170                                                      Sacox 60 ppm 101.0 95.0 94.1 98.2 100.0 9.8                                   Infection group                                                               F 100 577 34 55 1.5 605                                                       Sacox 60 ppm + 98.0 99.5 100.0 96.5 93.8 1.0                                  RBD-DHASCO 4%                                                                 Infection group                                                             __________________________________________________________________________     *kg Fl/kg LWI = kg of feed intake/kg of live weight increase             

What is claimed is:
 1. A method of preventing or treating parasiticdisease in animals comprising administering to an animal in needthereof, a pharmaceutically acceptable composition comprisingomega-3-fatty acid, obtainable from aquatic microorganisms.
 2. Themethod of claim 1, wherein the composition comprises biomass obtainableby cultivation of aquatic microorganisms.
 3. The method of claim 1,wherein the composition is an oil.
 4. The method of claim 1 wherein thecomposition comprises 40% DHA.
 5. The method of claim 4, wherein theaquatic microorganisms are heterotrophically cultivable microalgae. 6.The method of claim 5, wherein the microalgae belong to the orderCrypthecodinium.
 7. The method of claim 5, wherein the microalgae belongto the order Euglena.
 8. The method of claim 1, wherein the aquaticmicroorganisms are lower fungi.
 9. The method of claim 8, wherein thelower fungi belong to the order Thraustochytrium.
 10. The method ofclaim 8, wherein the lower fungi belong to the order Mortierella. 11.The method of claim 1, wherein the aquatic microorganisms arephotosynthesizing microalgae.
 12. The method of claim 11, whereinwherein the microalgae are selected from the group of orders consistingof Phaeodactylum, Isochrysis, Monodus, Porphyridium, Spirulina,Chlorella, Botryococcus, Cyclotella, Nitzschia, Dunaliella andNannochloropis.
 13. The method of claim 1, wherein the aquaticmicroorganisms are bacteria.
 14. The method of claim 13, wherein thebacteria are selected from the group of orders consisting of Alteromonasand Shewanella.
 15. The method of claim 1, wherein the composition isobtainable from mixtures of two or more different aquaticmicroorganisms.
 16. The method of claim 1, wherein the parasitic diseaseis avian coccidiosis.
 17. The method of claim 1, wherein the parasiticdisease is an endoparasitic disease of large or small animals.
 18. Apharmaceutical composition for prophylactic or therapeutic use against aparasitic disease in animals which comprises a disease preventing ortreating effective amount of an omega-3-fatty-acid-containingpreparation obtainable from aquatic microorganisms and at least onefurther coccidiostat.
 19. The composition of claim 18 further comprisingat least one other ingredient.
 20. The composition of claim 19, whereinsaid composition comprises salinomycin.
 21. The composition of claim 18,which comprises a biomass obtainable from aquatic microorganisms. 22.The composition of claim 18, which is an oil.
 23. The composition ofclaim 18, wherein said aquatic microorganisms are heterotrophicallycultivable microalgae.
 24. The composition of claim 23, wherein saidmicroalgae is from an order selected from the group consisting ofCrypthecodiniam and Euglena.
 25. The composition of claim 18, whereinthe aquatic microorganism is a lower fungus.
 26. The composition ofclaim 25, wherein said lower fungus belongs to the orderThraustochytrium or Mortierella.
 27. The composition of claim 18,wherein said microorganisms are photosynthesizing microalgae.
 28. Thecomposition of claim 27, wherein the microalgae are from an orderselected from the group of orders consisting of Phaeodactylum,Isochrysis, Monodus, Porphyridium, Spirulina, Chlorella, Botryococcus,Cyclotella, Nitzschia, Dunaliella and Nannochloropsis.
 29. Thecomposition of claim 18, wherein said microorganisms are bacteria. 30.The composition of claim 29, wherein said bacteria are from the orderAlteromonas or Shewanella.
 31. The composition of claim 18, wherein thepreparation is obtainable from one or more aquatic microorganisms.